Synergistic effect of sildenafil combined with controlled hypothermia to alleviate microglial activation after neonatal hypoxia–ischemia in rats

Background and purpose The only validated treatment to prevent brain damage associated with hypoxia–ischemia (HI) encephalopathy of the newborn is controlled hypothermia with limited benefits. Additional putative neuroprotective drug candidates include sildenafil citrate, a phosphodiesterase-type 5 inhibitor. The main objective of this preclinical study is to assess its ability to reduce HI-induced neuroinflammation, in particular through its potential effect on microglial activation. Methods HI was induced in P10 Sprague–Dawley rats by unilateral carotid permanent artery occlusion and hypoxia (HI) and treated by either hypothermia (HT) alone, Sildenafil (Sild) alone or combined treatment (SildHT). Lesion size and glial activation were analyzed by immunohistochemistry, qRT-PCR, and proteomic analyses performed at P13. Results None of the treatments was associated with a significant early reduction in lesion size 72h after HI, despite significant changes in tissue loss distribution. Significant reductions in both Iba1 + (within the ipsilateral hemisphere) and GFAP + cells (within the ipsilateral hippocampus) were observed in SildHT group, but not in the other treatment groups. In microglia-sorted cells, pro-inflammatory markers, i.e. Il1b, Il6, Nos2, and CD86 were significantly downregulated in SildHT treatment group only. These changes were restricted to the ipsilateral hemisphere, were not evidenced in sorted astrocytes, and were not sex dependent. Proteomic analyses in sorted microglia refined the pro-inflammatory effect of HI and confirmed a biologically relevant impact of SildHT on specific molecular pathways including genes related to neutrophilic functions. Conclusions Our findings suggest that Sildenafil combined with controlled hypothermia produces maximum effect in mitigating microglial activation induced by HI through complex proteomic regulation. The reduction of neuroinflammation induced by Sildenafil may represent an interesting therapeutic strategy for neonatal neuroprotection. Supplementary Information 111`The online version contains supplementary material available at 10.1186/s12974-024-03022-w.


Figure S2
: Quantification of cell-specific staining within segmented brain area, from a standardized threshold.Imaging processing for assessing immunostaining using the Fiji software in total hemisphere and four delimited brain regions: hippocampus, S1 cortex, perirhinal cortex and thalamus.
Quantifications were performed after images were converted into black and white 8-bit signal and according to a predefined standardized threshold.Then, each treatment was compared to untreated animals (HI) using a Kruskal-Wallis test followed by a Dunn's multiple comparison test when appropriate (#: p<0.05; ###: p<0.001; ####: p<0.0001).Table S1 : Primers sequences used for RT-qPCR.

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S7: GSEA analysis against reactome pathways looking for deregulated biological processes in the HI untreated group compared to the Sham group.
Table 19 20 S8: GSEA analysis against reactome pathways looking for deregulated biological processes in the HI+Hypothermia group compared to the HI untreated group.

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Table S9: GSEA analysis against reactome pathways looking for deregulated biological processes in the HI+Sildenafil group compared to the HI untreated group.
Table S10: GSEA analysis against reactome pathways looking for deregulated biological processes in the HI+Hypothermia+Sildenafil group compared to the HI untreated group.

Figure S3 :
Figure S3: Assessment of cell purity after magnetic antibody-based cell sorting.A: CD11b/c positive cells showed a large majority of Cx3CR1-positive cells and less than 1% astrocytic contamination.B: ACSA-1 positive cells showed a large proportion of GFAP positive astrocytes and less than 5% contamination from oligodendroglial lineage.RDC means recurrent DNA double-strand break clusters observed in neural stem/progenitor cells.

Figure S8 :
Figure S8: Gene expression polarization in GFAP-positive astrocytes sorted from the contralateral hemisphere of P13 rats subjected to HI at P11 and kept either on normothermia (HI, n=41) compared to sham (n=12), or treated by hypothermia alone (HT, n=19), Sildenafil ip alone (Sild, n=20), and the combined treatment (Sild+HT, n=18).Detailed gene expression of A1 (A) and A2 astrocytic markers (B).Quantified results are mean ± SD.Sham vs untreated (NT) HI animals were first compared using a non-parametric Mann Whitney t-test (**: p<0.01).Then, each treatment was compared to untreated animals (HI) using a Kruskal-Wallis test followed by a Dunn's multiple comparison test (all comparisons are not significant).

Figure S9 :
Figure S9: Over-representation analysis (ORA) using GeneOntology/biological process non redundant of the proteome of MG/M cells sorted from the ipsilateral hemisphere of P13 rats subjected to HI at P11 without and with neuroprotective treatment Sild+HT.This ORA analysis was performed to identify the enriched genesets inversely regulated by HI and in HI-injured animals treated by Sild+HT.GO pathways defined by logFC >1,3, p-value < 0.05 and FDR<0.25 were considered significantly up or downregulated.Four genesets were identified as inversely deregulated (highlighted in yellow). 10

Figure S10 :
Figure S10: Neutrophilic infiltration in the lesion site.

Table 15 16 S4 :
ORA analyses looking for deregulated biological processes in the HI group compared to the Sham group, depending on various the statistical thresholds.ORA analyses looking for deregulated biological processes in the HI+hypothermia group (HT) compared to the HI untreated group, depending on various the statistical thresholds. S3:

Table S5 :
ORA analyses looking for deregulated biological processes in the HI+Sildenafil group (Sild) compared to the HI untreated group, depending on various the statistical thresholds.

Table 17 S6:
ORA analyses looking for deregulated biological processes in the HI+Hypothermia+Sildenafil group (SildHT) compared to the HI untreated group, depending on various the statistical thresholds.